The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

Background Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences. Results About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein. An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the Chelonus lineage. Venom components specific to C. inanitus included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins. Conclusions The use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of C. inanitus appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.

Premature Osteoblast Clustering by Enamel Matrix Proteins Induces Osteoblast Differentiation through Up-Regulation of Connexin 43 and N-Cadherin

In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.

Glycomic analysis of human mast cells, eosinophils and basophils

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.

Proteome analysis in cardiovascular pathophysiology using Dahl rat model

Dahl salt-sensitive (DS) and salt-resistant (DR) inbred rat strains represent a well established animal model for cardiovascular research. Upon prolonged administration of high-salt-containing diet, DS rats develop systemic hypertension, and as a consequence they develop left ventricular hypertrophy, followed by heart failure. The aim of this work was to explore whether this animal model is suitable to identify biomarkers that characterize defined stages of cardiac pathophysiological conditions. The work had to be performed in two stages: in the first part proteomic differences that are attributable to the two separate rat lines (DS and DR) had to be established, and in the second part the process of development of heart failure due to feeding the rats with high-salt-containing diet has to be monitored. This work describes the results of the first stage, with the outcome of protein expression profiles of left ventricular tissues of DS and DR rats kept under low salt diet. Substantial extent of quantitative and qualitative expression differences between both strains of Dahl rats in heart tissue was detected. Using Principal Component Analysis, Linear Discriminant Analysis and other statistical means we have established sets of differentially expressed proteins, candidates for further molecular analysis of the heart failure mechanisms.

Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A-2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.

Microbial community analysis of Lake Chillisquaque, a small water system in Central Pennsylvania

Cyanobacteria are photosynthetic organisms that require the absorption of light for the completion of photosynthesis. Cyanobacteria can use a variety of wavelengths of light within thevisible light spectrum in order to harvest energy for this process. Many species of cyanobacteria have light-harvesting proteins that specialize in the absorption of a small range of wavelengths oflight along the visual light spectrum; others can undergo complementary chromatic adaptation and alter these light-harvesting proteins in order to absorb the wavelengths of light that are mostavailable in a given environment. This variation in light-harvesting phenotype across cyanobacteria leads to the utilization of environmental niches based on light wavelength availability. Furthermore, light attenuation along the water column in an aquatic system also leads to the formation of environmental niches throughout the vertical water column. In order to better understand these niches based on light wavelength availability, we studied the compositionof cyanobacterial genera at the surface and depth of Lake Chillisquaque at three time points throughout the year: September 2009, May 2010, and July 2010. We found that cyanobacterialgenera composition changes throughout the year as well as with physical location in the water column. Additionally, given the light attenuation noted throughout the Lake Chillisquaque, we are able to conclude that light is a major selective factor in the community composition of Lake Chillisquaque.

Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel β-strand interactions between enzyme and target is suggested.

Proteins encoded in genomic regions associated with immune-mediated disease physically interact and suggest underlying biology

Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these risk variants. It has previously been observed that different genes harboring causal mutations for the same Mendelian disease often physically interact. We sought to evaluate the degree to which this is true of genes within strongly associated loci in complex disease. Using sets of loci defined in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein-protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more densely connected than chance expectation. To confirm biological relevance, we show that the components of the networks tend to be expressed in similar tissues relevant to the phenotypes in question, suggesting the network indicates common underlying processes perturbed by risk loci. Furthermore, we show that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non-immune traits to assess its applicability to complex traits in general. We find that genes in loci associated to height and lipid levels assemble into significantly connected networks but did not detect excess connectivity among Type 2 Diabetes (T2D) loci beyond chance. Taken together, our results constitute evidence that, for many of the complex diseases studied here, common genetic associations implicate regions encoding proteins that physically interact in a preferential manner, in line with observations in Mendelian disease.

Comparison of genomic and proteomic data in recurrent airway obstruction affected horses using Ingenuity Pathway Analysis(R)

BACKGROUND: Recurrent airway obstruction (RAO) is a severe chronic respiratory disease affecting horses worldwide, though mostly in the Northern hemisphere. Environmental as well as genetic factors strongly influence the course and prognosis of the disease. Research has been focused on characterization of immunologic factors contributing to inflammatory responses, on genetic linkage analysis, and, more recently, on proteomic analysis of airway secretions from affected horses. The goal of this study was to investigate the interactions between eight candidate genes previously identified in a genetic linkage study and proteins expressed in bronchoalveolar lavage fluid (BALF) collected from healthy and RAO-affected horses. The analysis was carried out with Ingenuity Pathway Analysis(R) bioinformatics software. RESULTS: The gene with the greatest number of indirect interactions with the set of proteins identified is Interleukin 4 Receptor (IL-4R), whose protein has also been detected in BALF. Interleukin 21 receptor and chemokine (C-C motif) ligand 24 also showed a large number of interactions with the group of detected proteins. Protein products of other genes like that of SOCS5, revealed direct interactions with the IL-4R protein. The interacting proteins NOD2, RPS6KA5 and FOXP3 found in several pathways are reported regulators of the NFkappaB pathway. CONCLUSIONS: The pathways generated with IL-4R highlight possible important intracellular signaling cascades implicating, for instance, NFkappaB. Furthermore, the proposed interaction between SOCS5 and IL-4R could explain how different genes can lead to identical clinical RAO phenotypes, as observed in two Swiss Warmblood half sibling families because these proteins interact upstream of an important cascade where they may act as a functional unit.

Molecular analysis of carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) as a candidate gene for bovine spongiform encephalopathy susceptibility

Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP(C) into the pathogenic PrP(d). Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.

Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.

Comparative human-horse sequence analysis of the CYP3A subfamily gene cluster

Cytochrome P450 enzymes (CYP450s) represent a superfamily of haem-thiolate proteins. CYP450s are most abundant in the liver, a major site of drug metabolism, and play key roles in the metabolism of a variety of substrates, including drugs and environmental contaminants. Interaction of two or more different drugs with the same enzyme can account for adverse effects and failure of therapy. Human CYP3A4 metabolizes about 50% of all known drugs, but little is known about the orthologous CYP450s in horses. We report here the genomic organization of the equine CYP3A gene cluster as well as a comparative analysis with the human CYP3A gene cluster. The equine CYP450 genes of the 3A family are located on ECA 13 between 6.97-7.53 Mb, in a region syntenic to HSA 7 99.05-99.35 Mb. Seven potential, closely linked equine CYP3A genes were found, in contrast to only four genes in the human genome. RNA was isolated from an equine liver sample, and the approximately 1.5-kb coding sequence of six CYP3A genes could be amplified by RT-PCR. Sequencing of the RT-PCR products revealed numerous hitherto unknown single nucleotide polymorphisms (SNPs) in these six CYP3A genes, and one 6-bp deletion compared to the reference sequence (EquCab2.0). The presence of the variants was confirmed in a sample of genomic DNA from the same horse. In conclusion, orthologous genes for the CYP3A family exist in horses, but their number differs from those of the human CYP3A gene family. CYP450 genes of the same family show high homology within and between mammalian species, but can be highly polymorphic.

Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, beta-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry-based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.